Biocompatibility of gutta-percha solvents using in vitromammalian test-systemDaniel A. Ribeiro, DDS, PhD,a Mariza A. Matsumoto, DDS, PhD,b

Mariângela E. A. Marques, MD, PhD,c and Daisy M. F. Salvadori, BMD, PhD,c Botucatu andBauru, BrazilSÃO PAULO STATE UNIVERSITY AND UNIVERSITY OF SACRED HEART

Objectives. Taking into consideration that DNA damage and cellular death play important roles duringcarcinogenesis, the purpose of the present study was to evaluate in vitro genotoxic or cytotoxic effects of chloroformand eucalyptol by single cell gel (comet) assay and trypan blue exclusion test, respectively.Study design. Chloroform and eucalyptol were exposed to Chinese hamster ovary cells in culture directly for 3 hoursat 37°C at final concentrations ranging from 1.25 to 10 �L/mL. The negative control group was treated with vehiclecontrol (phosphate-buffered solution), and the positive control group was treated with methyl metasulfonate (MMS, at1 �g/mL concentration). All data were analyzed by the Kruskal-Wallis nonparametric test followed by the Dunn test.Results. The results showed that both gutta-percha solvents were cytotoxic at concentrations of 2.5, 5, and 10 �L/mL(P � .05). On the other hand, both solvents did not induce DNA breakage at 1.25 �L/mL concentration.Conclusions. These results suggest that both chloroform or eucalyptol are strong cytotoxicants, but they may not be afactor that increases the level of DNA lesions in mammalian cells. (Oral Surg Oral Med Oral Pathol Oral Radiol

Endod 2007;103:e106-e109)

Carcinogenesis is a multistep process characterized bygenetic, epigenetic, and phenotypic changes.1 Suchchanges involve genetic damage, mutation in criticalgenes related to the control of cell division, cell death,and metastatic potential, and activation of signalizingor metabolic pathways that give the cells favorablegrowth and survival characteristics.2 With increasingknowledge of these mechanisms, and the conclusionthat most cases of cancer are preventable, efforts havefocused on identifying the agents and exposures thatcause cancer.3

Gutta-percha has been used in endodontics for morethan 100 years and is today the most frequently usedcore material for permanent obturation of root canals.4

In cases of endodontic failure, however, endodonticretreatment must consist of the removal of gutta-perchacones from inside root canals. There are several meth-ods for gutta-percha removal: thermal, mechanical, andchemical. Chloroform is one of chemical agents widely

Supported by FAPESP (Fundação de Amparo à Pesquisa do Estadode São Paulo), (grant number 05/52567-8).aDepartment of Health Sciences, Federal University of São Paulo.bDepartment of Dental Clinics, University of Sacred Heart.cDepartment of Pathology, Botucatu Medical School, São Paulo StateUniversity.Received for publication Jun 9, 2006; returned for revision Sep 26,2006; accepted for publication Nov 16, 2006.1079-2104/$ - see front matter© 2007 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2006.11.024

e106

used for gutta-percha softening or dissolving. However,side effects from exposure to chloroform have beenreported in literature.5 Among them, studies have ad-dressed that the chloroform is possibly carcinogenic tohumans.6,7 Because of the concerns about the chloro-form, clinicians and researchers have developed a re-newed interest in finding alternative solvents.8-10 Euca-lyptol, a widely used substance for flavoring andfragrance, has been used as a solvent without apparentharmful effects.11-13 Taking into consideration thatDNA damage and cellular death play important rolesduring carcinogenesis, studies on genotoxic or cyto-toxic effects of chloroform and other gutta-percha sol-vents, such as eucalyptol, are needed for the compre-hensive risk assessment of these compounds. For thisreason, the purpose of the present study was to evaluatein vitro genotoxic and cytotoxic effects of chloroformand eucalyptol.

To evaluate the magnitude of DNA damage, thesingle cell gel (comet) assay was chosen. This is arapid, simple, and reliable biochemical technique forevaluating DNA damage in mammalian cells.14 Thebasic principle of the single cell gel (comet) assay is themigration of DNA fragments in an agarose matrixunder electrophoresis. When viewed under a micro-scope, cells have the appearance of a comet, with ahead (the nuclear region) and a tail containing DNAfragments or strands migrating toward the anode.15 Tomonitor cytotoxic effects, trypan blue assay was ap-

plied in this setting.

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MATERIALS AND METHODSCell culture

Chinese hamster ovary (CHO K-1) cells were growthto confluence in 75-mL culture flasks (Corning NewYork, Corning, NY) using Ham’s F-10 medium (In-vitrogen Corporation, Grand Island, NY) supplementedwith 10% fetal calf serum and 100 U/mL penicillin(Life Technologies, Carlsbad, CA) and 100 �g/mLstreptomycin (Invitrogen Corporation) at 37°C with 5%CO2. Cells were cultured for 5 days before treatmentwith the test substances. Confluent cells were detachedwith 0.15% trypsin (Invitrogen Corporation) for 5 min-utes, then 2 mL complete medium was added and cellswere centrifuged at 1000 rpm (180 g) for 5 minutes.Cell suspension was counted using a Neubauer cham-ber and seeded in 96-well microtiter plates (Corning) ata density of 1 � 104 cells per well (at a concentrationof 1 � 106/mL).

Treatment of cellsThe materials used were chloroform (Merck, Darm-

stadt, Germany) and eucalyptol (Inodon Laboratório,São Paulo, Brazil). All materials tested were preparedin increasing final concentrations ranging from 1.25�L/mL to 10 �L/mL. The negative control group wastreated with vehicle control (phosphate-buffered solu-tion, PBS) and the positive control group was treatedwith methyl metasulfonate (MMS, at 1 �g/mL; SigmaAldrich, St. Louis, MO), an alkylating agent able toinduce DNA breakage. After incubating a volume of 20�L of treated or control cells (1 � 104 cells) for 3 hoursat 37°C, the cells were centrifuged at 1000 rpm (180 g)for 5 minutes, washed twice with fresh medium, andresuspended with fresh medium. Each individual treat-ment was repeated in 3 separate experiments in differ-ent days to ensure reproducibility.

Cytotoxicity assayCytotoxicity was performed using trypan blue stain-

ing after the treatment.16 In brief, a freshly preparedsolution of 10 �L trypan blue (0.05%) in distilled waterwas mixed to 10 �L of each cellular suspension for 5minutes, spread onto a microscope slide, and coveredwith a coverslip. Nonviable cells appear blue. At least200 cells were counted per treatment.

Genotoxicity assayThe protocol used for single cell gel (comet) assay

followed the guidelines purposed by Tice et al.14

Briefly, a volume of 10 �L of treated or control cellswas added to 120 �L 0.5% low-melting-point agaroseat 37°C, layered onto a precoated slide with 1.5%regular agarose, and covered with a coverslip. After

brief agarose solidification in a refrigerator, the cover-

slip was removed and slides immersed in lysis solution(2.5 mol/L NaCl, 100 mmol/L EDTA (Merck), 10mmol/L Tris-HCl buffer pH 10 (Sigma Aldrich), and1% sodium sarcosinate (Sigma Aldrich), with 1% Tri-ton X-100 (Sigma Aldrich) and 10% DMSO (Merck))for about 1 hour. Before electrophoresis, the slides wereleft in alkaline buffer (0.3 mmol/L NaOH (Merck) and1 mmol/L EDTA (Merck), pH �13) for 20 min andelectrophoresed for another 20 minutes at 25 V (0.86V/cm) and 300 mA. After electrophoresis, the slideswere neutralized in 0.4 mol/L Tris-HCl (pH 7.5), fixedin absolute ethanol, and stored at room temperatureuntil analysis blindly in a fluorescence microscope at400� magnification. An automated analysis system(Comet Assay II; Perceptive Instruments, Haverhill,United Kingdom) was used to determine DNA damageby tail moment (product of tail DNA/total DNA by thecenter of gravity) from 50 cells selected randomly pertreatment.17 To minimize extraneous DNA damagefrom ambient ultraviolet radiation, all steps were per-formed with reduced illumination.

Statistical methodsData of 3 independent repeats from the comet assay

and the cytotoxicity assay were assessed by theKruskal-Wallis nonparametric test followed by theDunn test, using SigmaStat software, version 1.0 (JadelScientific, Chicago, IL). The level of statistical signif-icance was set at 5%.

RESULTSThe cytotoxicity of gutta-percha solvents was mea-

sured in CHO cells through trypan blue assay in arange-finding experiment before the determination ofchemically induced genotoxicity. The percentage ofviable cells exposed to chloroform or eucalyptol isshown in Fig. 1. At concentrations of 10, 5, and 2.5�L/mL, the percentage of cell viability for both gutta-percha solvents tested were statistically significant(P � .05) compared with negative control in a dose-related fashion. However, at the dilution of 1.25 �L/mL,no statistically significant differences (P � .05) weredetected, and more viable cells were found in the chloro-form than in eucalyptol.

The single cell gel (comet) assay was used to mea-sure DNA damage in CHO cells in vitro. DNA strandbreaks were represented by the mean tail moment for50 comets/sample. As seen in Table I, both compoundsevaluated did not induce strand breaks in DNA at1.25 �L/mL concentration.

DISCUSSIONIntroduction of chemicals in the working environ-

ment requires the assessment of their harmful effects.

OOOOEe108 Ribeiro et al. May 2007

Progress in biocompatibility in vitro tests concerningcytotoxicity and/or genotoxicity of gutta-percha sol-vents has been minimal over the past decade, and fewinvestigations in the field of dental research have takenfull advantage of the many end point assays that havebeen developed in the general field to investigate dif-ferent compounds. The trypan blue exclusion test canbe used to indicate cytotoxicity, where dead cells takeup the blue stain of trypan blue and live cells haveyellow nuclei. Cytotoxicity data obtained in our labo-ratory on CHO cultures demonstrated that chloroformwas able to produce cellular death in a dose-relatedfashion, the strongest effect being observed at higherconcentrations. This is consistent with published datareporting that chloroform is a strong cytotoxicant inseveral cellular types.18-20 In the same way, eucalyptolinduced cellular death compared with negative controlat higher concentrations. A recent study conducted byZaccaro Scelza et al.20 showed that eucalyptol was

Fig. 1. Effects of serial concentrations of chloroform andeucalyptol on trypan blue exclusion test. Results are ex-pressed as the mean percentage of control (mean � SD).�P � .05 compared with negative control (zero).

Table I. Mean � SD of DNA damage (tail moment) inChinese hamster ovary cells exposed to gutta-perchasolventsSolvent tested (1.25 �L/mL) DNA damage

Negative controla 0.43 � 0.21Chloroform 0.60 � 0.20Eucalyptol 0.70 � 0.16Positive controlb 4.23 � 0.94*

aPhosphate-buffered solution (pH 7.4).bMMS at 1 �g/mL.*P � .05 compared with negative control.

cytotoxic in a cell culture assay. On the basis of present

data, we assumed that both solvents tested are able tobind to cell membrane and readily penetrate the cells,leading to lethal cellular injury. Therefore, both gutta-percha solvents evaluated are cytotoxins, and eucalytolpresented more pronounced effect.

The alkaline version of the single cell gel (comet)assay was used here to identify putative genotoxins.This assay is sensitive for a wide variety of DNAlesions. Among them are DNA strand breaks, alkali-labile sites, lesions including abasic sites, and incom-plete repair sites.14 It shows clear advantages in itsapplicability to almost all cell types. Limitations, how-ever, need to be accepted concerning the validity ofdetecting genotoxic effects in situations with elevatedcytotoxic effects. Because cytotoxicity produces strandbreaks that show up as increased DNA migration, it isnot recommended to perform the comet assay on sam-ples with more than 30% cytotoxicity.14 Therefore, thecomet assay was conducted for both gutta-percha sol-vents at the lowest concentration only. The resultsclearly demonstrated that chloroform did not contributeto damage to DNA. Our results are in line with otherreports.19,21 Moreover, studies have provided evidencethat tumor induction by chloroform occurs via non-genotoxic-cytotoxic mode of action.22,23 Eucalyptol didnot induce DNA breakage. Previous studies have con-firmed that eucalyptol is not considered a potentialgenotoxin.24 For a more detailed judgment on the geno-toxic potential of gutta-percha solvents, however, abattery of genotoxicity tests is needed.

In summary, results presented here support the con-clusion that both chloroform or eucalyptol are strongcytotoxicants. Therefore, continuous exposure to thesecompounds are of special concern, because cytotoxicityis considered to be a prime mechanism in nongenotoxicmechanisms of carcinogenesis. Regarding genotoxicitydata, both gutta percha solvents may not be a factor thatincreases the level of DNA lesions in mammalian cellsfor the lowest concentration tested. Nevertheless, thisfinding should be interpreted cautiously, because theconcentration in clinical usage is far higher than thisconcentration (1.25 �L/mL).

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Reprint requests:

Daniel A. Ribeiro, DDS, PhDDepartamento de Ciências da Saúde, Universidade Federal de SãoPauloUNIFESPAv Ana Costa 9511060-001Santos, SP, Brazil

daribeiro@unifesp.br